Spinning apparatus for measurement of characteristics relating to molecules

ABSTRACT

An apparatus for measuring a characteristic of a sample using a centrifuge and optical components is disclosed. The centrifuge may be a standard benchtop centrifuge. The optical components may be sized and dimensioned to fit, along with the sample, inside the centrifuge.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofInternational Application PCT/US2017/019318, filed Feb. 24, 2017, whichwas published under PCT Article 21(2) in English, and which claims thebenefit of U.S. Provisional Application No. 62/299,711, entitled“SPINNING APPARATUS FOR MEASUREMENT OF CHARACTERISTICS RELATING TOMOLECULES,” filed on Feb. 25, 2016. These applications are incorporatedherein by reference.

FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. Government support under grant number2011080983 awarded by the National Science Foundation and grant number1R21GM107907-01A1 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

BACKGROUND 1. Field

Aspects described herein relate generally to a spinning apparatus formeasurement of characteristics relating to molecules, and methods ofmeasuring such characteristics.

2. Discussion of Related Art

The ability to quantify interactions between biomolecules is of greatinterest for scientific and medical research, as well as for drugdevelopment. Examples of measurable characteristics of a biomolecularinteraction include the affinity (e.g., how strongly the moleculesbind/interact) and the kinetics (e.g., rates at which the associationand dissociation of molecules occur) of the interaction. Traditionally,such characteristics are measured in solution, using methods such ascalorimetry, stop-flow imaging, or surface plasmon resonance. These bulkmeasurements are limited in many ways, including 1) they report onlyaverage behavior and thus may lose important details associated withmetastable states and rare events, and 2) they measure chemistry in theabsence of externally applied mechanical stress, which can bedramatically different from crowded and dynamic environments in livingsystems.

Force probes that apply single molecule measurement methods includeatomic force microscopes (AFM), optical traps, magnetic tweezers,biomembrane force probes, and flow chambers. Due to technicalcomplexities, some systems require a large investment of money and time(e.g. optical trap systems typically cost S150,000 or more).Additionally, molecular interactions are studied one molecule at a timein most cases. Statistical characterization of these interactions istherefore slow and painstaking, requiring hundreds or thousands ofmeasurements which are typically performed in a serial manner.

SUMMARY

In an illustrative embodiment, an apparatus for measuring acharacteristic of a sample device is provided. The apparatus includes amodule, which includes a sample holder, a light source configured toilluminate the sample, and a detector configured to receive light fromthe sample. The module is sized and dimensioned to fit within acentrifuge receptacle having a volume of less than or equal to 1 L.

In another illustrative embodiment, a method includes attaching aparticle to a surface through a molecular interaction associated with afirst molecule and a second molecule. The method also includes rotatingthe surface about an axis of rotation to apply a centrifugal force tothe particle, the centrifugal force having a direction. The methodfurther includes hitting the particle with light from a light source anddetecting an image of the particle with a detector during rotation ofthe surface, the image containing information representing acharacteristic of the molecular interaction. The method also includesdetermining the characteristic of the molecular interaction based on thedetected image. An imaging axis is angled relative to direction ofcentrifugal force, the imaging axis being oriented along the directionat which light from the light source hits the sample.

In yet another illustrative embodiment, a method includes attaching aDNA nanoswitch to a surface and rotating the surface a first time toapply a force to the particle. After rotating the surface the firsttime, the method includes stopping rotation of the surface. Afterstopping rotation of the surface, the method further includes rotatingthe surface a second time to apply a force to the particle. The methodalso includes detecting images of the particle with a detector duringrotation of the surface the first time and the second time, the imagescontaining information representing a characteristic of the molecularinteraction. The method further includes determining the characteristicof the molecular interaction based on the detected images.

In yet a further illustrative embodiment, a method includes attaching aparticle to a surface through a molecular interaction associated with afirst molecule and a second molecule. The method also includes insertingthe surface into a centrifuge and selecting a centrifuge temperatureusing a temperature control built into the centrifuge. The methodfurther includes rotating the surface a first time to apply a force tothe particle and detecting an image of the particle with a detectorduring rotation of the surface, the image containing informationrepresenting a characteristic of the molecular interaction. The methodalso includes determining the characteristic of the molecularinteraction based on the detected image.

Various embodiments provide certain advantages. Not all embodiments ofthe present disclosure share the same advantages and those that do maynot share them under all circumstances.

Further features and advantages of the present disclosure, as well asthe structure of various embodiments are described in detail below withreference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments will now be described, by way of example, withreference to the accompanying drawings, in which:

FIG. 1A depicts a simplified schematic representation of an apparatusfor measuring a characteristic of a sample;

FIG. 1B depicts a centrifugal force applied to the sample of FIG. 1A;

FIG. 2 depicts a schematic of one embodiment of an apparatus formeasuring a characteristic of a sample;

FIG. 3 depicts a top-down view into a centrifuge of a spinning forcesystem;

FIG. 4 depicts a detailed view of a slip ring of the centrifuge of FIG.3 with a cable running through the slip ring;

FIG. 5 depicts a cutaway view of a module held within a centrifugebucket (left) and a centrifuge bucket with the module removed (left);

FIG. 6 depicts a perspective view of the module of FIG. 4;

FIG. 7 depicts an exploded view of the module of FIG. 4;

FIG. 8 depicts a typical field of view during a force spectroscopyexperiment using the spinning force apparatus;

FIG. 9 depicts a schematic of a measurement method according to oneembodiment;

FIG. 10 depicts a graph associated with the method depicted in FIG. 9,the graph showing tracking resolution of tether extension for variousbucket angles;

FIG. 11 depicts a graph angle associated with the method depicted inFIG. 9, the graph showing the minimum tether length, scaled by the beadradius, required to measure tether extension from lateral displacement,calculated as a function of the bucket;

FIG. 12 depicts a schematic of a method of measuring the bucket angleassociated with different rotational velocities;

FIG. 13 depicts data from one experiment using the method depicted inFIG. 9;

FIG. 14 depicts a schematic of a DNA nanoswitch construct in a loopedand an unlooped state, and data associated therewith;

FIG. 15 depicts graphs associated with repeated rupture forcemeasurement of single molecular pairs;

FIG. 16 depicts graphs associated with parallel DNA force-extension andoverstretching measurements made with a spinning force system;

FIG. 17 depicts images associated with verification of the DNA unzippingnanoswitch construct;

FIG. 18 depicts a schematic of a DNA nanoswitch construct beingsubjected to the method shown in FIG. 9 and associated data graphs; and

FIG. 19 details the force and loading rate of a spinning force system.

DETAILED DESCRIPTION

The system and methods of operation discussed herein can providemassively-parallel high-throughput single-molecule force measurements ata low cost. More specifically, rotation-induced forces (e.g.,centrifugal forces and viscous drag forces) can be used to manipulateevents such as single molecules (e.g., proteins or DNAs), singlemolecular interactions or molecular complexes (e.g., receptor-ligandprotein pairs), enabling forces to be applied to many eventssimultaneously and/or repeatedly. Each event (also referred to herein asa subject) can be observed directly and independently for truesingle-molecule detection. The efficiency of experiments can beimproved, reducing the time to conduct an experiment from days tominutes. More than simply speeding up experiments, this efficiency alsoenables new experiments such as near equilibrium measurements thatobserve interactions with hour-long lifetimes, which would be unfeasiblewith sequentially collected statistics. Furthermore, with largerstatistical sets more easily attainable, more detailedcharacterizations, model testing, and observations of populationheterogeneity are possible. Parallel measurements can also be used totest families of interactions simultaneously (e.g., multiple drugcandidates could be tested simultaneously against a target receptor).

The system and methods of operation discussed herein can provideaccurate force control in a wide range of directions and magnitudes.Through force control, the system and methods of operation can be usedto quantify force dependent interactions, including measuring the forcedependence of kinetic parameters (e.g., K_(on) and K_(off)) andmolecular subtleties which would be invisible from population averaging.Using this system, the mechanical properties of biomolecular complexes(e.g., compliances of DNAs and proteins) and cellular targets (e.g.,elasticity of stress-bearing cells) can be studied, yielding valuableinformation into both the structure and the function of those subjects.

The centrifugal force field applied to a sample in some embodiments ofthe system and methods of operation described herein is macroscopicallyuniform, stable without the need for active feedback, calibration-free,and dynamically controllable in an essentially deterministic way. Thus,a desired force history can be applied to an ensemble (or plurality) ofsingle molecules or events (whether identical or different from eachother) without the need for active feedback. The force fieldconveniently couples to mass density, eliminating the possibility ofradiative damage and expanding the range of systems that can be studiedwith force (e.g., beads or objects made of any material can be used, aslong as they have a different mass density than their surroundings).Furthermore, by varying the bead type, bead size, and rotation speed, awide range of forces, at least from sub-femtoNewtons to nanoNewtons, canbe achieved.

The system and methods of operation described herein can be convenientlyintegrated with various types of force probes to generate forces inmultiple dimensions with high flexibility. For example, the system canbe used in conjunction with optical traps, magnetic tweezers and/ormicrofluidic devices to generate a combination of forces (such asgradient and scattering forces, magnetic forces, hydrodynamic forces,and centrifugal forces). Each force can be applied to a sample (orevent) in a different direction, with a different magnitude, and/or at adifferent test stage.

The system and methods of operation described herein can also beconveniently integrated with various imaging techniques to providereal-time observation with high temporal and spatial resolution. Forexample, using interference techniques and diffraction analysis, theposition of individual particles in a sample can be ascertained withsub-nanometer accuracy. Also, fluorescent imaging can be used to enablevisualization of subtle molecular transitions during experiments.Moreover, using video tracking by high-speed CCD cameras, molecularevents can be detected on the scale of microseconds.

In some embodiments, the systems and methods described herein can bemore cost effective and simpler to use than other common methods ofmolecular spectroscopy. The material cost of a module described hereinis generally less than the cost of a typical laboratory microscope. Insome embodiments, experiments using this system are straightforward,with a pre-preprogrammed force protocol, minimal setup, and little or noneed for user intervention.

The inventors have appreciated that the ability to mechanicallymanipulate single molecules or molecular interactions can lead toinsights throughout biomedical research, from the action of molecularmotors in replication and transcription to the role of mechanical forcesin development. The inventors have recognized that, while in principlethese approaches enable the full characterization of individualmolecular complexes and the study of population heterogeneity at thesingle-molecule level, in practice, key challenges exist. The firstchallenge for force spectroscopy studies is the low throughput of mostsingle-molecule approaches. Furthermore, sufficient statistics must becollected not only for the population, but also for each individualmolecule or interaction or complex, which can be a challenge forstudying catastrophic transitions such as bond rupture. Anotherchallenge is the positive identification of the single-moleculeinteractions of interest over non-specific and multiple interactions.Finally, there is the subtle challenge of noise, both thermal andexperimental, that makes distinguishing different populations ofmolecules with similar force properties difficult.

The inventors have addressed these challenges with spatiotemporallymultiplexed force spectroscopy. In some embodiments, the system canaccomplish parallel spatial multiplexing with repeated interrogation. Insome embodiments, repeated interrogation is enabled by self-assemblednanoscale devices. The inventors have developed a spinning force systemfor high-throughput single-molecule (or single interaction)experimentation that, in some embodiments, utilizes a commercialbenchtop centrifuge.

In the spinning force system, an entire microscope imaging system isrotated to observe microscopic objects subjected to uniform centrifugalforce (unlike earlier “spinning disk” centrifuge microscopes^(19, 20)).This design enables, in some embodiments, temperature control andhigh-resolution particle tracking (˜2 nm). As an illustrative embodimentand application, the inventors have developed a high-throughput assaythat integrates mechanical nanoswitches, such as those described inpublished PCT application WO2013/067489 (the entire contents of whichare incorporated herein) to provide important new functionality. Thenanoswitches can serve two roles—one as a molecular signature tofacilitate reliable and automated analysis of large data sets, and thesecond to enable the repeated interrogation of each single-molecule pair(or single molecular interaction), increasing throughput and enablingnew measurements of heterogeneity in single-molecule (or singlemolecular interaction) experiments. By making repeated forcemeasurements on hundreds of single-molecule complexes (such as singlenanoswitches that, as described below, may embrace both members of abinding pair), multiple statistics on each molecule (or nanoswitch, andthus each interaction of interest) that comprises the population can becollected. The inventors have also recognized that by averaging multiplerupture forces on a per-molecule basis (e.g., per nanoswitch basis),noise can be reduced to enable super-resolved force spectroscopy—theidentification of different populations of molecules (e.g.,nanoswitches) below the thermal force-resolution limit. Averaging allowsreduction of the spread in force distributions (averaging reduces noiseby a factor of ˜sqrt(N)) without losing information about differencesbetween molecules (e.g., nanoswitches). Furthermore, the rich andrelatively large data sets provided by this technique could alsocomplement other analysis techniques for statisticaldeconvolution^(22, 23).

Schematic Overview

Referring to FIGS. 1A and 1B, when rotary arm 120 is in operation,sample 140 rotates at an angular velocity ω and at a distance R from thecenter of axis 102. For illustrative purposes, in this example, sample140 includes a cover slip 140′ having an inner surface 140 a and anouter surface 8 with respect to axis 102. Both surfaces are aligned inparallel with axis 102. A particle 240 (e.g., a bead) adheres to outersurface 8 through a chemical bond 243 formed between molecule A 242 andmolecule B 244. In this example, molecule A is a receptor chemicallylinked to outer surface 8, and molecule B is a ligand chemically linkedto particle 240. (The techniques and methods for forming such linkageswill be described in greater detail below).

When particle 240 undergoes circular motion, a centrifugal force F isexerted on the particle, as defined by the following equation:

$\begin{matrix}{F = \frac{{mv}^{2}}{R}} & (1)\end{matrix}$

where F is the net centrifugal force, m and v are the mass and thelinear velocity of the particle, respectively, and R is the distance ofthe particle from rotation axis 102. In a rotating reference frame inwhich orbiting particle 240 appears stationary, particle 240 experiencesan inertial centrifugal force equal to F in a direction perpendicular toouter surface 140 b and away from central axis 102 (shown by arrow 250).In some examples where particle 240 is a spherical bead in solution withradius r and relative density ρ, rewriting equation (1) in terms ofangular velocity ω yields:

$\begin{matrix}{F = \frac{4{\pi\rho}\; r^{3}R\;\omega^{2}}{3}} & (2)\end{matrix}$

When sample 140 rotates about axis 102 at a very low speed, centrifugalforce F is countered by the interaction force of chemical bond 243,allowing particle 240 to continue to adhere to surface 8. As the angularvelocity ω rises, the increasing magnitude of centrifugal force F causesbead 240 to move with respect to surface 8. The characteristics of therelative motion (e.g., the root-mean-square displacement or thedirection of the motion) can be monitored and analyzed to quantifycertain chemical and/or mechanical properties of bond 243 (e.g.,properties associated with its transitional states and conformationalchanges). The increasing F may also cause the rupture of chemical bond243, at which point, particle 240 is released from surface 8. Themagnitude of centrifugal force F at the particle release indicates therupture force of chemical bond 243.

During operation, the magnitude of centrifugal force F can becontrolled, for example, by adjusting the angular velocity ω of rotaryarm 120. For instance, sample 140 can be subjected to multiple cycles offorce application in which the centrifugal force on particle 240 isincreased and/or decreased through step changes in angular velocity ω.

In addition to changing the angular velocity ω, it is also possible tochange the radius of rotation R either statically or dynamically bychanging the sample position relative to the axis of rotation. Forexample, in system 100, sample 140 may be mounted to an adjustablerotary arm with extendible length, or staged on a positioner that can betranslated in a radial direction with respect to central axis 102.

In embodiments where particle 240 is a spherical bead, the centrifugalforce F can also be varied by changing one or more of the particlecharacteristics ρ and r shown in equation (2). For example, microspheresare commercially available in a wide range of materials and sizes (seeTable 1 below). By conjugating subjects of study (e.g., molecules,proteins, nucleic acids, cells, etc.) to selected beads, such asmicrospheres, the centrifugal force applied to the beads (and translatedto the subject) can be varied based on bead properties. In addition, thedegree of monodispersity of beads can control the range of forcesapplied for a given spin. For instance, a highly monodisperse sample(e.g., using beads of substantially the same size and properties) maycause all beads to experience the same force, while a polydispersesample (e.g., using beads of various sizes and/or properties) would havea wide range of forces being applied. Moreover, ρ of particle 240 canalso be altered by changing the density of the buffer solution.Furthermore, the geometry of the sample chamber can be varied to controlthe effects of fluid flow, which can add hydrodynamic forces toimmobilized particles in the chamber.

TABLE 1 Materials and sizes of beads Specific Density Size Range BeadMaterial (g/cm³) (μm) Borosilicate 1.5   1-100+ Polystyrene 0.05 0.05-100+ Silica 1.2 0.01-100  Gold 18.3 0.002-0.25  Melamine 0.510.5-10  Iron Oxide 4.24 10-Jan

With proper parameter selection, the force applied to particle 240 canspan 9 orders of magnitude, ranging from microNewtons (e.g., r=10 μm,ρ=1.5 g/cm³, R=500 mm, ω=100 Hz) to femtoNewtons (e.g., r=1 μm, ρ=0.05g/cm³, R=250 mm, ω=2 Hz).

The direction of the centrifugal force F can also be controlled. In someexamples, sample 140 may be coupled to a surface 8 (e.g., of a coverglass, other coverslip or other sample chamber or sample mountingelement) that is perpendicular to rotational axis 102, resulting in acentrifugal force F along surface 8. In some embodiments, sample 140 maybe coupled to an outer surface 8 (i.e., a surface facing away from therotational axis 102) that is parallel to rotational axis 102, resultingin a tensile force on the bond 243, as shown in FIG. 1B. For particularimplementations, it may be desirable to couple a sample to a surfacethat is parallel to rotation axis 102 because pulling particle 240 awayfrom surface 8 reduces the likelihood of the particle forming newinteractions with unoccupied binding sites of molecule A on the surface8. In some embodiments, a compressive (rather than tensile) force can beapplied to bond 240 if the sample is coupled to an inner surface 140 a(i.e., a surface facing toward the rotational axis 102).

In some embodiments, sample may be coupled to a surface that forms aselected angle with respect to rotational axis 102 so that centrifugalforce F may be applied in any given direction. In some implementations,it may be desirable to couple sample to a surface that is at an obliqueangle with respect to the rotation axis 102. As will be discussed in alater section, this oblique angle arrangement may allow one to tracklateral particle motion—i.e., motion of the particles occurring parallelto the surface to which the sample is coupled.

In addition to centrifugal force F, other types of forces can also beapplied to particle 240 through spinning. For example, if particle 240is contained in a chamber filled with a liquid medium, the rotation ofsample 140 can generate regional flows that exert a viscous drag forceto particle 240. The direction of the drag force depends on factors suchas the geometry of the chamber and the orientation of the sample. Themagnitude of the drag force depends on factors such as the viscosity andthe temperature of the liquid medium, the size of the particle, and theangular velocity and acceleration of the sample.

In the apparatus for measuring a characteristic of a sample (such as amolecule such as a nanoswitch), motion of particle 240 (e.g.,displacement caused by molecular folding, unfolding or rupture of bond243) can be observed by video tracking methods (e.g., by takingsuccessive images of the particle at a high temporal resolution). Aswill be discussed, a light source, sample and objective may rotatetogether at the same angular velocity ω, and thus these three componentsappear stationary to each other in a rotating reference frame.Therefore, images of particle 240 can be formed using traditionalimaging techniques, including transmitted- or reflected-light techniquesand fluorescence techniques.

System Overview

According to one aspect, in some embodiments, the apparatus formeasuring a characteristic of a sample is a small-scale spinning forcesystem that uses a centrifuge that is standard equipment in scientificlaboratories. In some embodiments, the centrifuge is a benchtopcentrifuge, such as the Thermo Scientific Heraeus X1R Centrifuge. Theuse of standard equipment can decrease the cost and increase theaccessibility of the spinning force approach to molecularcharacterization.

The spinning force system also includes a module that, in someembodiments, holds the optical components and the sample beinginvestigated. The module can be sized and shaped to fit within thecentrifuge, e.g., within a bucket of the centrifuge or within the holesor slots of the centrifuge. In some embodiments, the module is sized andshaped to fit within a 400 mL bucket volume or less. In someembodiments, the module is smaller, and is sized and shaped to fitwithin a 15 mL, 50 mL or less, 100 mL or less or 1 L or lesstube/bucket/volume.

Referring to panel A of FIG. 2, a spinning force system 100 uses astandard laboratory centrifuge 30 to provide rotational force for thestudy of molecular interactions. In some embodiments, the module 300that holds the optical components 390 and the sample 400 may be sized tofit within one of the swing buckets 40 of the centrifuge. The opticalcomponents 390 may include a detector 21, an objective 11, and a lightsource 22. A battery 345 for powering the optical components and/or amedia converter may be included inside the centrifuge bucket 40. Thespinning force system 100 may include a computer 1419 that receivessignals from the detector 21. In some embodiments, signals from thedetector 21 are first sent to a media converter, which then sendssignals to a computer 1419. In some embodiments, the computer 1419 orother controller may be arranged to control the centrifuge, e.g.,control angular velocity, angle of the bucket, rotation duration, startand stop times, etc. The centrifuge may use a control module thatenables control by a computer or other controller.

The module's sample holder may be a chamber, slot or other space orattachment point in the module into which sample or something holdingthe sample can be inserted or otherwise attached to. For example, aswill be discussed in more detail below, in some embodiments, a samplechamber 260 comprises two glass surfaces adhered together (e.g. at theirborders), while leaving a gap between the coverslips within the bordersto accommodate sample. Sample, such as a population of nanoswitches, iscoupled to one of the surfaces and portions of the sample are permittedto move toward or away from the surface it is coupled to in response toan application of centrifugal force from the centrifuge. The samplechamber can then be inserted into the module's sample holder, which maybe a slot or space into which the sample chamber may be snapped into,slid into, or otherwise coupled to. In some embodiments, the surface towhich the sample is coupled to (e.g., the cover glass or othercoverslip) may be disposable.

A variety of different types of samples may be held by the sampleholder. In some embodiments, the sample includes nanoswitches, such asDNA nanoswitches. Panel B of FIG. 2 depicts a magnified view of thesample 400 that is coupled to a surface 8 of a sample chamber 260, whichis in turn held by a sample holder of a module (an example of a sampleholder is shown as item 340 in FIGS. 5 and 7). In some embodiments, thesample may be directly coupled to the sample holder of the module, suchthat no sample chamber is needed.

The sample 400 in FIG. 2 includes a plurality of nanoswitches such asDNA nanoswitches. The images corresponding to t0, t1, and t2 depict theconfiguration of the nanoswitches over time, as the applied force isincreased over time, e.g. due to increased angular velocity of thecentrifuge. The graph at the lower portion of panel b reflects tetherextension monitored as a function of time when force is applied.Molecular transitions such as a bond rupture between a receptor(R)—ligand (L) pair, the pair being part of the nanoswitch, causes awell-defined change in tether extension of the nanoswitch, providing adistinct signature for detecting interactions between two molecules ofinterest, both of which may be integral to the nanoswitch.

Thus, the methods and devices can be used to measure interactionstrength or other parameter relating to two separable and physicallydistinct binding partners that are bound to each other and then areseparated from each other via a bond rupture. One such binding partneris conjugated to a detectable moiety such as a particle and it is thelocation of the detectable moiety that denotes information about thestate of the bond between the two binding partners.

In the case of a nanoswitch, both binding partners are conjugated to orare part of the same molecule (such as for example a nucleic acid),referred to herein as the nanoswitch. The binding partners are spacedsufficiently apart from each other along the nanoswitch such that theyare able to interact and bind to each other. The nanoswitch comprises adetectable moiety such as a particle, at or near its free end.Increasing the force on the nanoswitch, via the particle for example,will rupture the bond between the two binding partners, thereby causingthe length of the nanoswitch to change (i.e., increase, typically), andin so doing the position of the particle also changes.

FIG. 2, panel B depicts some nanoswitches in an open or extendedconformation and other nanoswitches in a closed or looped conformation.Those in the open or extended conformation have experienced a bondrupture, and this informs an end user that the force applied tonanoswitch is greater than the binding force (or energy) between the twobinding partners. Conversely, those in the closed or looped conformationhave not yet experienced a bond rupture, and this informs an end userthat the force applied to the nanoswitch is less than the binding force(or energy) between the two binding partners.

The various nanoswitches on a support in a single run may be identicalto each other or they may be different. Identical nanoswitches comprisethe same backbone molecule and the same binding partners at the samepositions along the backbone molecule (e.g., nucleic acid). Differentnanoparticles may comprise different binding partners, optionally atdifferent positions along the backbone molecule (e.g., nucleic acid). Ifat different positions, then different nanoswitches, and thus differentbinding pairs, may be identified based on the change in their lengthfrom a looped to an extended conformation.

The above description assumes that a nanoswitch comprises two bindingpartners and is used to study the binding characteristics of two bindingpartners to each other. However, it should be understood that thisdisclosure contemplates more complex nanoswitches that involve two ormore binding pairs and/or three or more binding partners that interactand/or potentially compete for binding with each other.

One illustrative example of a spinning force system 100 is shown in FIG.3, which depicts a top-down view into a centrifuge 30 having fourbuckets. The centrifuge may be a standard benchtop centrifuge that iscommonly found in scientific laboratories (e.g., the Thermo ScientificHeraeus X1R Centrifuge). The first bucket 40 holds a module containingoptical components and a sample. In some embodiments, transmission ofcamera data out of the centrifuge during centrifugation is accomplishedby converting the camera's gigabit Ethernet signal to a fiber-opticsignal, then passing this data out of the centrifuge through a fiberrotary joint. The adjacent bucket 41 holds electrical components forsignal conversion. In one embodiment, the second bucket 41 includes amedia converter 57 that converts camera signals from the module to afiber optic signal. The bucket also includes a battery to power themedia converter 57. A first cable 37 connects the module 300 to themedia converter 57. In some embodiments, the media converter is part ofthe module. A second cable 39 is a fiber optic cable that sends a fiberoptic signal from the media converter out to a computer or other deviceoutside of the centrifuge. In some embodiments, the fiber optic cablemay run through a slip ring 38 before exiting the centrifuge. FIG. 4shows a detailed view of the slip ring 38 with the cable 39 runningthrough it. As is well known in the art, the slip ring is anelectromechanical device that allows the transmission of power and/orelectrical signals from a stationary to a rotating structure. The slipring 38 allows the portion of cable downstream of the slip ring (i.e.the portion of the cable exiting the centrifuge) to remain stationarywhile the centrifuge rotor and the cable upstream of the slip ring (i.e.the portion of cable running from the media converter to the slip ring)rotates.

In an embodiment using the Thermo Scientific Heraeus X1R Centrifuge, theTX-400 rotor can be modified to mount a fiber optic rotary joint(Princetel MJX) along the central axis. This can be accomplished byremoving the rotor's central push-release mechanism (by loosening thescrew on the side of the button), and threading the four existingthrough holes to accept 10-32 screws. An adapter can be installed on therotor to hold the slip ring. In one illustrative example, FIG. 4 depictsan adaptor 36 that is installed to accommodate the slip ring 38. Toenable the fiber optic cable to pass through the lid of the centrifuge,the central plastic viewing window of the centrifuge was removed.

It should be appreciated that, in some embodiments, the electricalcomponents for converting signals may be incorporated into the module,or may be eliminated completely. For example, in some embodiments, themodule may send signals wirelessly such that no conversion to a fiberoptic signal is needed. Turning back to FIG. 3, the next two buckets 42,43 hold balances 35 that balance out the weight of the first twobuckets.

The module will be discussed in detail next. The left side of FIG. 5shows that the module 300 is sized to fit within a centrifuge bucket 40.The right side of FIG. 5 shows the centrifuge bucket with the moduleremoved. The module may hold optical components as well as the sample400. The optical components of the module include a light source in theform of an LED, an objective, a detector 21 in the form of a camera, anda turning mirror that redirects light from the light source 22 to thecamera. To allow the module to fit within a confined volume, in someembodiments, the optical path may include two right-angle bends. In theillustrative embodiment of FIG. 5, the turning mirrors 18 enable thesetwo right-angle bends. As a result, some of the optical components maybe placed side-by-side to one another rather than being placed in astraight line. In this manner, the overall length of the entire assemblyis decreased, enabling the module to be compact enough to fit within thecentrifuge bucket.

In one illustrative embodiment shown in FIGS. 5-7, a red LED (Thorlabs,LED630E) threaded to a tube mount (Thorlabs, S1LEDM) served as the lightsource. A glass diffuser positioned between the LED and sample chamberprovided uniform illumination across the field of view. The 25 mmdiameter of the sample chamber was designed to be compatible with theSM1 lens tube. The sample was magnified and imaged onto a CCD camera(AVT, Prosilica, GC 2450) with a 40× Olympus Plan Achromat objective(infinity corrected, 0.65 NA and 0.6 mm WD) and Ø1″ 100 mm tube lens(Thorlab, AC254-100-A). The camera used the standard GigE Visioninterface, outputting the data as a gigabit Ethernet signal. To enablelive imaging during centrifugation, a fiber-optic rotary joint(PrinceTel, MJX) can be installed at the center of the centrifuge rotor.The camera signal can be converted from twisted-pair Ethernet to afiber-optic signal by a small media converter inside of the centrifuge,transferred through the rotary joint, then converted back to a standardEthernet signal by a second media converter connected to the acquisitioncomputer. In one illustrative example, the MiniMc Gigabit products, fromIMC Networks, (e.g., part numbers 855-10734 and 855-10735) can serve asthe media converters. The images collected from the detector can berecorded.

To measure sample temperature, in one embodiment, a portable wirelessthermocouple connector (Omega Engineering, MWTC-D-K-915) may be embeddedwith a surface adhesive thermocouple (Omega Engineering, SA1XL-K) withinthe bucket that contains the module. A wireless receiver (e.g., OmegaEngineering, WTC-REC1-915) can be used to acquire the temperature fromthe thermocouple connector to record the temperature in real time.

FIG. 6 depicts shows a perspective view of the module alone, without thecentrifuge bucket. As can be seen from FIG. 6, the module has a U-shapedarrangement. The first leg 301 and the second leg 302 of the U-shapeneed not be the same length. In the embodiment shown in FIG. 6, thefirst leg 301 is longer than the second leg 302. In the illustrativeembodiment of FIG. 6, the light source 22, objective 11 and sampleholder 340 are located within the first leg 301, and the detector 21 islocated within the second leg 302. However, it should be understood thatthe optical components and sample holder may be arranged in any orderand placed in either leg.

It should be appreciated that, in other embodiments, the light source,objective, sample and detector can be aligned, thus eliminating the needfor a turning mirror. For example, in larger centrifuges, such as largerfloor models with 1 Liter buckets, the greater bucket depth may fit thelight source, objective, sample and detector in a line without needingto bend the optical path.

In yet other embodiments, the optical path may have one or more bends atangles other than right angles, e.g., the optical path may have a 15,30, 45, 60 or 85 degree bend. Such bends may be accomplished using oneor more mirrors.

In some embodiments, the module includes a housing 305 that secures thecomponents of the module and ensures a tight fit within the bucket orother volume within the centrifuge. The housing may include an open slotfor a battery (e.g., SparkFun, PRT-00339), and a connected DC-to-DC stepup circuit (e.g., SparkFun, PRT-08290) to serve as the power source forthe light source, detector, and, in some embodiments, media converter.The housing 305 may be 3D printed, injection molded, die cast, or formedby any other suitable method. In some embodiments, the housing is madeof acrylonitrile butadiene styrene (ABS) was.

FIG. 7 depicts an exploded view of one illustrative embodiment of themodule 300. A list of parts corresponding to each numerical referencenumber is listed in Table 2 below. It should be appreciated that thislist of parts is only one illustrative embodiment, and that othersuitable parts may be interchanged with those on the list.

TABLE 2 Parts List of Module Reference Part No. Name Vendor NumberDescription 1 Light source Thorlabs S1LEDM SM1-Threaded mount Mount 2Coupler Thorlabs SM1T1 SM1 (1.035″- 40) Coupler 3 Retaining ringThorlabs SM05RR for diffuser 4 Light source Thorlabs DG05- Ø½″ N-BK7diffuser 220 Ground Glass 5 Diffuser and Thorlabs SM1A6T first half ofsample holder 6 Support glass - SI Howard D265 Ø 25 mm, first side ofGlass Co 0.7 mm Thick sample chamber assembly 7 Double sided Kapton TapePPTDE-1 tape holding the two glasses (6, 8) together 8 Cover glass -Electron 63782-01 Gold Seal, #1 second side of Microscopy 19 mm sampleSciences chamber assembly 9 Second half Thorlabs SM1L03 SM1 Lens ofsample Tube, 0.3″ holder Thread Depth 10 Focusing lens Thorlabs SM1V05Focusing Ø1″ tube SM1 Lens Tube 11 Objective Edmund #86-815 40× OlympusOptics Plan Achromat Objective, 0.65 NA, 0.6 mm WD 12 Objective ThorlabsSM1A3 Objective Adapter Adapter with External SM1 Threads and InternalRMS Threads 13 Achromatic Thorlabs AC254- f = 100.0 mm, doublet 100 Ø1″Achromatic Doublet, ARC: 400-700 nm 14 Tube lens Thorlabs SM1RR TubeLens SM1 retaining ring Retaining Ring 15 Objective lens Thorlabs SM1M20Objective SM1 tube Lens Tube Without External Threads, 2″ Long 16Adapter Thorlabs SM1A6T Adapter with External SM1 Threads and InternalSM05 Threads, 0.40″ Thick 17 Housing for Aluminum turning mirrors 18Turning Thorlabs PFE10- 1″ Silver mirrors P01 Elliptical Mirrors, 450nm-20 μm 19 Camera Thorlabs SM1NT Camera SM1 locking ring (1.035″-40)Locking Ring, Ø1.25″ Outer Diameter 20 Camera Thorlabs SM1A9 Cameraadapter Adapter with External C- Mount Threads and Internal SM1 Threads21 Detector Allied Vision Prosilica Sony ICX625 Technologies GC2450 CCDsensor, 2448 × 2050 resolution, 15 fps, 12 bit 22 Light source ThorlabsLED630E Red LED

In the illustrative embodiment of FIG. 7, parts 5 and 6 combine to formthe sample holder 340, and parts 6-8 combine to form the sample chamber260. Part 5 can have two functions—it forms part of the sample holderand acts as a diffuser. The sample chamber is held within the sampleholder. The turning mirrors 18 are held by a housing 17 that connectsthe mirrors to the rest of the assembly and holds the mirrors at aproper angle. The housing 17 may also serve to connect the first leg ofthe U-shape to the second leg.

The detector used in the illustrative embodiment of FIG. 7 is equippedwith a 5 Megapixel CCD sensor with a maximum frame rate of 15 fps atfull resolution. Depending on the application, alternative cameras withdifferent resolutions and acquisition rates could be used as thedetector—for example, the Basler Ace CMOS camera at 15 Megapixels or 2Megapixel version is under $400 (Edmund Optics, Inc.) The mediaconverter (item 22) that converts the twisted wire Ethernet connectionto an optical signal could be omitted if a camera with a 10 GigE opticalfiber output is used. Data output using a compact wireless router is analternative approach, but may result in a slower acquisition rate.Additional customized parts such as the turning mirror housing can bereplaced with a Thorlabs compact cage cube system. In some cases, asolid aluminum construction may provide added stability of the imagingpath.

In some embodiments, the detector may be a wireless video camera, suchas an action camera (e.g. GoPro HERO) or a cell phone. The camera can bewirelessly controlled and can wirelessly stream and/or record at fullresolution. Such an arrangement would avoid the need to run cablesthrough the centrifuge. In some cases, such an arrangement would alsoavoid the need for a media converter.

FIG. 8 depicts a typical field of view during a force spectroscopyexperiment using the spinning force apparatus, showing that thousands ofsurface-tethered beads can be monitored in parallel.

Sample Chamber Construct

In one illustrative embodiment, the sample chamber can be constructedusing double-sided Kapton tape sandwiched between a 25 mm diametersupport glass and a 19 mm diameter cover glass (Gold Seal, 3346). Two 1mm diameter ports, which serve as a solution inlet and outlet, aredrilled into a 0.7 mm thick support glass (S.I. Howard Glass (D263)).The cover and support glasses can be cleaned by immersing in 100 mL a 1%(v/v) Hellmanex III solution, microwaving for 1 minute, then sonicatingfor 30 minutes. Subsequently, the slides may be rinsed thoroughly withMillipore water then dried with nitrogen flow. A 1 mm×7 mm rectangularflow channel is cut on the double-sided Kapton tape using a cut plotter(Graphtec). To form tethers with digoxigenin functionalized construct,the cover glass is functionalized with anti-digoxigenin using a modifiedversion of a previously developed protocol³². First, the cover glass iscoated with a nitrocellulose solution by depositing 2 μL of amyl acetatesolution with 0.2% (m/v) dissolved nitrocellulose. The channel is thenincubated with phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl,10 mM phosphate buffer, pH 7.4) solution containing 100 ug/mlanti-digoxigenin (Roche, 11333089001) for 15 minutes. The channel isthen washed and further incubated with a surface passivation solution(10 mg/ml Roche Blocking Reagent in PBS) for 1 hour. After thepassivation step, the channel is flushed with experimental buffer thenincubated with 5 pM of construct for 15 minutes. At 5 pM constructconcentration, the construct may be limited to an average spacing ofroughly 2 μm on the surface, which may make formation of double tethersa rare event. After tethering the construct to the surface, the flowchannel is washed with 20 uL of the experimental buffer then incubatedwith 15 mg/ml streptavidin beads (Invitrogen M-270). For eachexperiment, beads can be washed excessively with the experimental bufferbefore loading them to the sample chamber. Before loading the samplechamber into the module of the spinning force system, the solution inletand outlet ports are sealed with vacuum grease. The Tris experimentalbuffer may consist of 10 mM Tris, 30 mM NaCl at pH 7.5 with or without10 mM MgCl₂.

For the overstretching experiment, the surface tethering wasstrengthened by replacing digoxigenin-anti-digoxigenin withbiotin-streptavidin—in other words, biotin-streptavidin interactionswere used to anchor each tether. The nitrocellulose surface wasfunctionalized by incubating it with 1 mg/ml streptavidin in PBSsolution that contained 1 mg/ml of Roche Blocking Reagent for 12 hour,followed by incubation with passivation solution (10 mg/ml RocheBlocking Reagent in PBS buffer) for 1 hour. The channel was flushed withPBS and incubated with 5 pM of dual-biotin λ-DNA for 15 minutes beforeloading in the streptavidin-coated beads. Under such conditions, thedensity of streptavidin on the surface was sparse enough that only oneend of the biotin-labelled λ-DNA bound to the surface, leaving the otherbiotinylated end free to bind to the streptavidin-coated bead.

Angled Measurement Method

According to one aspect, the inventors have developed a method ofmeasuring nanometer-level extensions of tethers in the spinning forcesystem by projecting tether length changes onto the X-Y plane of thesurface to which sample is coupled (e.g. a cover glass or othercoverslip), enabling these measurements to be made in a relativelysimple and computationally efficient way. The method utilizes the factthat the direction of force application by the centrifuge can becontrolled by mechanically constraining the angle of the centrifugebucket or, in centrifuges without buckets, the holder(s) in thecentrifuge that holds sample (e.g. holes into which test tubes or othersample holding containers are inserted). In the method, the direction ofcentrifugal force and the imaging axis are intentionally misaligned.

The imaging axis is oriented in the direction along the direction inwhich light from the light source passes through or is otherwiseincident to the sample. For example, if the optical components andsample are in a straight line, the imaging axis is defined as the axisalong that line. If the optical components and sample are not in astraight line, and devices are used to redirect light, (e.g., mirrors),the imaging axis is oriented along the direction in which light from thelight source hits the sample. In the U-shaped configuration of theembodiment of FIGS. 4-6, the imaging axis is the line along which thelight source, sample, and objective are aligned.

In some embodiments, a spinning force system is arranged such that thedirection of force and the imaging axis are intentionally misaligned inorder to track tether extension length by tracking lateral particlemotion—i.e., motion of the particle occurring perpendicular to theimaging axis. When the direction of force and the imaging axis arealigned, particles move only in a direction parallel to the imagingaxis. In such a situation, change in tether length is difficult to trackbecause all that the detector sees is the tethered particle (e.g., bead)getting larger in size or smaller in size.

When the direction of force and the imaging axis are intentionallymisaligned, the detector sees lateral movement of the particle. Thisinformation can be used to determine tether extension length.

Based on the geometry as illustrated in FIG. 9, extension of themolecular tether can be measured by tracking the motion of the tetheredparticle 240 (e.g., a bead) parallel to the surface to which sample iscoupled (e.g. a cover glass or other coverslip). Specifically, changesin tether extension will appear to the detector as a lateraldisplacement of the bead ΔL_(obs). The actual changes in tetherextension ΔL in the direction parallel to the force can be calculatedbased on the angle of the bucket θ relative to the rotation axis 102 asfollows:

$\begin{matrix}{{\Delta\; L} = \frac{\Delta\; L_{obs}}{\cos\;(\theta)}} & (3)\end{matrix}$

The minimum tether length that can be measured using this method dependson both the bead size used in the experiment and the angle of thecentrifuge bucket relative to the rotation axis. If the tether length isnot long enough to allow the bead to be pulled away from the surface 8,the bead may end up making direct contact with the surface 8, which willresult in inaccurate measurements. The minimum tether length L_(min) asa function of bead radius R_(bead) and bucket angle θ is given by:

$\begin{matrix}{L_{\min} = {R_{bead}\left( {\frac{1}{\sin(\theta)} - 1} \right)}} & (4)\end{matrix}$

FIG. 11 shows the minimum tether length L_(min), scaled by the beadradius, required to measure tether extension from lateral displacement,calculated as a function of the bucket angle θ.

The angle of the bucket θ relative to the rotation axis can bedetermined in different ways, depending on the type of centrifuge thatis used. With centrifuges having a single fixed angle such as a fixedangle rotor centrifuge or a vertical rotor centrifuge, the angle θ isknown, as it does not change. With centrifuges that change angle withchanging rotation speed, such as a swinging-bucket rotor, measurementsmay be needed to determine the angle of the bucket θ associated withdifferent angular velocities.

In one exemplary method, illustrated in FIG. 12, a marker or othermarking device may be attached to the bottom of a bucket. The markermarks the height of the bucket bottom on the wall as the centrifugespins. The vertical distance a from the pivot point 49 to the mark 50and the horizontal distance b from the pivot point 49 to the mark 50 aremeasured. With these known distances, the angle of the bucket relativeto the rotation axis is given by:θ=a tan(b/a)  (5)

In one experiment using the arrangement of FIG. 12, the uncertainty ofthe angle measurement was based on the distance measurement errorestimate of 1 mm. At the angular velocity of 300 RPM, the bucket swungout to an angle of (81.4±0.8)°. At a much higher speed of 1800 RPM theangle increased by 2.3%. Further increase of the angular velocity didnot increase the angle beyond the error of the measurement.

FIG. 10 shows tracking resolution of tether extension for bucket anglesbetween 20° to 85° from one example using this method. Using the angledmeasurement method, tether length resolution can be “tuned” based on thebucket/sample holder angle with a range of approximately 2.5 nm (at) 20°to 12 nm (at 80°) based on a lateral tracking resolution of ˜2 nm. Datafrom one example using this angled measurement method is shown in FIG.13. The drift-corrected X and Y position of a 5 um silica bead as afunction of time recorded in the centrifuge spinning at 2,000 RPM isshown as the zigzagging line. The drift in X and Y position as afunction of time, based on the average position of 12 immobile referencebeads, is shown as the smoother trend curve. The panels to the rightshow histograms of the position with normal distribution fits. Standarddeviations of the fits for x and y positions are both ˜2 nm. While thestandard deviation listed here represents the particle-trackingresolution, the overall accuracy with which tether lengths can bemeasured also depends on the intrinsic thermal fluctuations of thesebeads.

To validate and demonstrate this angled measurement method, DNAforce-extension for over 100 molecules were measured simultaneously overa span of less than 1 minute, and each were fit with the standardworm-like chain model. The most likely contour length and persistencelength was 8.2±0.2 μm and 46±1 nm, respectively, in agreement withexpected values²⁴. Additionally, multiplexed overstretching measurementsof lambda DNA were performed, yielding an overstretching force of63.5±1.7 pN (mean±SD), consistent with previous measurements at theseconditions²⁴.

FIG. 16 shows parallel DNA force-extension and overstretchingmeasurements made with a spinning force system. Panel A of FIG. 16depicts force-extension data of half lambda DNA (24 kbp) obtained from asingle sample with 113 DNA tethers using a centrifuge bucket constrainedto a 20° angle. The top panel shows a scatter plot of the persistencelength and contour length obtained from fits to the worm-like chainmodel performed for each tether (n=113). Histograms projecting thepersistence length and contour length of the model onto the x- andy-axis, respectively, are shown as straight lines indicating expectedvalues. The bottom panel shows the force-extension curves ofsingle-tethered DNA data filtered by persistence length and contourlength (n=30). Ranges were selected from the peaks of the histograms(one bin-width on either side), yielding filtering ranges of 43-48 nmand 7.8-8.6 μm for the persistence length and contour length,respectively. The overlaid curve represents the expected force-extensioncurve.

Panel B of FIG. 16 depicts multiplexed DNA overstretching measured withthe spinning force system. The top panel shows a representativeforce-extension curve near the overstretching transition. Theoverstretching force was extracted as the half-way-point of the twooverstretching transition forces and is shown as a vertical line betweenthe dashed vertical lines. The bottom panel shows a histogram of theoverstretching force measured from a single sample (n=29) with anaverage and standard deviation of 63.5±1.7 pN.

Nanoswitches for Authenticating Single-Molecule Data

According to one aspect, nanoswitches such as nucleic acid nanoswitchesare used with the spinning force system. The inventors have recognizedthat the use of nucleic acid nanoswitches, such as DNA nanoswitches,with the spinning force system can help to enable robust and repeatablerupture experiments. In some embodiments, these molecular switches aredesigned to adopt a looped structure when the molecules of interest areinteracting and a linear structure when they are not, as seen in FIG.14, panel A. As a result, nanoswitches provide a distinct “signature”(i.e. increase in tether length) for rupture events, as seen in FIG. 17,panel B.

In one example, DNA unzipping experiments were performed on a 29 bp DNAinteraction, and the “signature” unlooping of the nanoswitches was usedto positively identify and discriminate valid single-molecule data frommultiple tethers and non-specific interactions.

Panels A of FIG. 14 depicts a schematic of a nanoswitch in a looped andan unlooped state. Two complementary oligos hybridize to form a loopednanoswitch. Force can unzip the two complementary strands, resulting ina measurable increase in tether length, providing a signature of DNAunzipping.

Panel B of FIG. 14 depicts images of a bead tethered to the surface viaa nanoswitch showing the looped and unlooped states. The scale bar is 1μm long.

Panel C of FIG. 14 depicts one example of rupture force measurement. 381tethers were identified with the nanoswitch transitions signature tocollect rupture forces while the remaining 673 transitions thatcorresponded to bead detachment and improper transitions were omitted.

Panel D of FIG. 14 depicts unzipping force histograms of 29 bp dsDNAmeasured with the nanoswitch under two different buffer conditions.Unzipping force measurements were carried out in the presence andabsence of magnesium ions, with hundreds of rupture statistics for eachcondition collected in under 30 seconds of centrifuge run time.Magnesium was found to stabilize the duplex, with the average unzippingforce (±the standard deviation) increasing from 10.1±0.9 pN to 14.6±1.1pN with the addition of magnesium.

Controllable Temperature Conditions

The inventors have recognized that it can be desirable to conductexperiments in different controlled temperatures. The inventors havealso recognized that many standard centrifuges have built-in temperaturecontrol and/or portability to move into cold (e.g., 4° C.) or warm(e.g., 37° C.) rooms. The inventors have appreciated that using thesekinds of centrifuges in the spinning force system permits experimentswith temperature control.

As an example, DNA unzipping experiments were performed using a spinningforce system having a centrifuge with built-in temperature control atfour temperatures, 4, 13, 23, and 37° C. Panel E of FIG. 14 depicts theaverage unzipping force of 29 bp dsDNA under different temperatures withPBS buffer (Total n=306), with histograms of rupture forces shown as aninset. The theoretical line is calculated using a previously describedthermodynamic model²⁹.

Real-time measurements of the sample temperature during experiments weremade with a wireless thermocouple embedded within the centrifuge bucket.An increase in the unzipping force with decreasing temperature wasobserved.

Repeated Interrogation and Super-Resolved Force Spectroscopy

The spinning force system and nanoswitches can be used to repeatedlyinterrogate a population of molecules (or interactions) at thesingle-molecule (or single interaction) level. In one example, FIG. 15depicts graphs associated with repeated rupture force measurement ofsingle molecular pairs.

Panel A of FIG. 15 depicts a protocol for repeated cycles of forceapplication, with each cycle consisting of a linear force ramp to inducerupture and nanoswitch unlooping, followed by a low force reassociationperiod allowing the molecular pairs to rebind.

Panel B of FIG. 15 depicts a DNA unzipping force histogram of 1863rupture events collected from a total of 538 molecular pairs with 12cycles of force application. The gradient from darkest to lightestcorresponds to statistics collected from each cycle. This datademonstrates the large amounts of single-molecule force data that can beaccumulated with this approach.

The nanoswitches enable the unique properties of each molecule in asample to be characterized from repeated measurements, as demonstratedby determining a rupture-force histogram for each molecule in a sample,illuminating population heterogeneity at the single-molecule level.Panel C of FIG. 15 depicts exemplary rupture force histograms generatedfor individual molecular pairs. The calculated average rupture force isshown below the graphs.

Furthermore, by averaging data from multiple pulls of the same molecularpair, the spread in force is reduced without losing the uniquecharacteristics of each molecule. When applied to data from a singlepopulation, this per-molecule (or per interaction) force averaginggenerates a super-resolved histogram with the expected narrowing whencompared to the raw histogram of all the data. Panel D of FIG. 15depicts a combined histogram of rupture forces from 27 molecules with 7cycles of force rupture each (top), and histogram of the per-moleculeaveraged rupture force (bottom), showing a reduced width.

When applied to combined statistics from two populations of DNA zippers(introducing another G-C rich zipper with a higher unzipping force³⁰),the super-resolved histogram generated from per-molecule averaging canseparate out two populations that are unresolveable from the rawhistograms due to the intrinsic broadening of force that results fromthermal noise and instrumental noise. Panel E of FIG. 15 depicts acombined histogram for two populations of DNA unzipping experiments(top), and the per-molecule averaged super-resolved histogram (bottom)that recovers the two separate populations from the mixed data.

Multiple rupture events can be collected for each molecule in a set byrepeatedly spinning the same sample multiple times. For the datapresented in FIG. 15, panel B, the sample was spun up with an effectiveforce loading rate of 1 pN/s to a maximum force of 22 pN. The speed wasthen ramped down to zero rpm for approximately 1 hour to allow rebindingbetween each pair of molecules. Beads were identified from cycle tocycle by their positions relative to a set of fiducial beads which werecommon to each cycle. For the measurement of the two populations ofmolecules, two different DNA nanoswitch unzipping constructs were made,one with 48% GC content (CACGAATTCTCTGCCTCCCTTTTAACCCTAG, SEQ ID NO: 1)and one with 31% GC content (CTCAAATATCAAACCCTCAATCAATATCT, SEQ ID NO:2).

Example of a Nanoswitch Construct Method

In some embodiments, looped nanoswitches, such as DNA nanoswitches, canbe made according to the following exemplary process. Circular M13mp18singlestranded DNA (ssDNA) (New England Biolabs, N4040S) was linearizedby hybridizing a 40 bp oligo that created a double-stranded restrictionsite for the BtsCI enzyme (New England Biolab, R0647S). Subsequently, aset of complementary oligos (Integrated DNA Technologies) was hybridizedonto the linear ssDNA. Functionalized oligos (biotinylated anddigoxigenin-modified) were hybridized onto the 3′ and 5′ ends of thessDNA respectively. The hybridization was carried out with 15 nM oflinearized ssDNA and 10 molar excess of the complementary oligos in 1×NEBuffer 2 with a temperature ramp from 90 to 20° C. (−1° C./minute) ina thermocycler. After this initial hybridization two specificsingle-stranded regions remained, which were bridged by twopartially-complimentary oligos to form the final looped construct. Thesequence of the complimentary bridge oligo that formed the loop was:CTCAAATATCAAACCCTCAATCAATATCT, SEQ ID NO: 2. This secondaryhybridization step was carried out at a final construct concentration of250 pM with a 1.25 molar excess of the bridge oligos in 1× NEbuffer 2 atroom temperature for 1 hour.

Looping of the construct was verified using gel-shift assays,single-molecule optical trap measurements, and AFM imaging, as seen inFIG. 17. FIG. 17 depicts images associated with verification of the DNAunzipping nanoswitch construct. Panel A of FIG. 17 depicts gelelectrophoresis of the DNA nanoswitch showing loop formation. Lane i isthe 1 kbp extension ladder (Invitrogen, 10511-012), lane ii is a linearconstruct without the two complementary oligos that close the loop, andlane iii is the nanoswitch construct with the two complementary oligosthat can form the looped nanoswitch. The looped DNA migrates more slowlyin the gel than the linear construct, resulting in a discrete band witha higher apparent molecular weight as previously observed^(36,37).

Panel B of FIG. 17 depicts a force-extension curve of the DNA unzippingnanoswitch construct measured using optical tweezers. The line to theleft corresponds to the looped construct, and the line to the rightcorresponds to the unlooped construct. When forces above ˜12 pN wereapplied, the 29 bp dsDNA that formed the loop unzipped, causing thetether length to increase to the full length of the M13 dsDNA tether.

Panel C of FIG. 17 depicts AFM images of a DNA unzipping nanoswitchconstruct. The arrow indicates the putative location of the hybridizedDNA zipper. The length of the scale bar is 100 nm.

In the optical trap measurement, force was applied on the looped DNAconstruct via tethering between laser-trapped streptavidin andanti-digoxigenin functionalized silica beads.

For the DNA overstretching measurements in the spinning force system,both ends of lambda DNA was functionalized with biotin to provide stronganchorage to the streptavidin functionalized cover glass and beadsurfaces. First, 20 μL of lambda DNA (0.28 ug/ml, Roche, 10745782001)was incubated for 20 minutes at 65° C. to remove the hybridizedoverhangs. Subsequently, a nucleotide mixture that consists ofBiotin-14-dATP, Biotin-14-dCTP, dTTP and dGTP, each at 100 μM finalconcentration, was added to the lambda DNA solution with 0.25 U/mlKlenow Fragment (New England Biolabs, M0212S). This mixture wasincubated for 1 hour at 37° C. The dual-end biotin lambda DNA waspurified from the excess nucleotides and enzyme using the Qiagen PCRPurification Kit.

For the parallel force-extension measurements, the half-length lambdaDNA was made functionalized with digoxigenin and biotin. First, thebiotin-labeled full-lambda DNA construct was cut near the middle usingthe Xbal restriction enzyme (New England Biolab, R0145S). The resultingoverhangs were functionalized with digoxigenin to produce aheterobifunctional 24 kbp construct labeled with digoxigenin on one sideand biotin on the other.

Angled Measurement Method with Nucleic Acid Nanoswitch Constructs

When a nucleic acid nanoswitch construct, such as a DNA nanoswitch, isused with the angled measurement method discussed above, the loopopening signature can be identified by tracking the beads' X and Ypositions. With the imaging axis and the direction of centrifuge forcebeing at an angle, a component of the centrifugal force is directed inthe X-Y plane, as seen in FIG. 18, panel A. As the rotational speed ofthe centrifuge increases, the looped DNA tethers continuously extend.The opening of the loop can be identified as a discontinuous change inextension. Panel A of FIG. 18 depicts images of a bead before (above),and after (below) a loop opening transition. A bead which is tethered tothe surface with a single DNA nanoswitch will undergo a discontinuouschange in position with a well-defined length ΔL_(obs), and direction φ.

In one exemplary experiment, each movie taken contained approximately1000 beads. To track these beads, they were first identified using theMatlab function imfindcircles. A template image for each bead wasstored. To identify the bead in the subsequent frame, the template imagewas scanned in the X-Y plane to find the position of maximumcorrelation. First, the image was scanned in the X direction over a 25pixel search region centered on the bead position from the previousframe. A 2^(nd) order parabola was then fit to the correlationcoefficient as a function of position. The position of maximumcorrelation was identified as the new bead position. The template imagewas than centered on the new X position, and the same procedure was donein the Y direction. During the course of each experiment, there was somedrift in the X-Y plane. This was corrected for by taking the medianchange in X and Y for all beads being tracked from frame to frame. Thisdrift correction can be sufficient for identifying the looped toun-looped transition.

Panel B of FIG. 18 depicts a scatter plot of all contour length changesdetected for all directions. The gradient represents data density. Onlytransitions within the boxed region are accepted as nanoswitchtransitions. Inset, a histogram of transition forces for three differenttypes of transitions: beads which leave the surface (“beads detaching”),beads which display discontinuous transition with (“nanoswitchtransitions”) and without (“improper transitions”) correct direction andmagnitude. Panel C of FIG. 18 depicts a scatter plot of the symmetryratio and root mean square displacement based on the lateralfluctuations of all tracked beads, with the gradient representing datadensity. The overlaid black data points are for tethered beads thatundergo validated nanoswitch transitions.

As shown in FIG. 18, panels B and C, transitions were identified byfiltering out all bead trajectories except those that contained adiscontinuous change in extension of both the correct magnitude anddirection. This removed false transitions that may have resulted fromnon-specific interactions or the formation of multiple bonds between thebead and the surface. Following this automated filtering procedure, thetransition events were visually inspected, in random order, to rejectany remaining erroneous transitions, which may have occurred due toparticle tracking artifacts that manifest as discontinuous changes inposition (e.g., particle mislabeling, overlapping beads, etc.).

For DNA unzipping experiments, the number of transitions as a functionof time was converted to the number of transitions as a function offorce as follows: the rotational speed of the centrifuge was recordedduring each movie using WinMess software (provided by Thermo FisherScientific, R&D) which enables communication and control with thecomputer. The rotational speed was then converted to force (F) as afunction of time using the following equation:F=m _(bead) Rω ²  (6)where R and ω are the rotational radius and rotational velocity,respectively. To determine the effective mass of the beads (m_(bead)) insolution, the Invitrogen M-270 bead density was measured throughsink-float analysis using aqueous sodium polytungstate solution(Sigma-Aldrich, 71913). A density of 1.61±0.02 g/cm³ was obtained, whichthat confirmed the manufacturer's (Life Technologies) reported value of1.6 g/cm³. The manufacturer also provided the bead diameter of ourspecific lot giving a mean of 2.80 um with <1.6% CV (lot #144315600).The density of the silica beads used in the DNA force extension andoverstretching experiment was measured similarly using the sodiumpolytungstate solution, yielding a density of 1.50±0.03 g/cm³. Thediameter of the silica beads was measured using transmission electronmicroscopy (TEM), yielding an average and standard deviation of4.27±0.16 um.

FIG. 18 depicts a schematic of a nucleic acid nanoswitch construct, suchas a DNA nanoswitch, being subjected to the angled measurement methoddescribed above, in which the direction of force and the imaging axisare intentionally misaligned, e.g. the centrifuge bucket is at anoblique angle to the rotation axis and the surface to which the sampleis coupled is at an oblique angle to the rotation axis, the detectorsees lateral movement of the particle. The centrifuge bucket is at anangle θ relative to the rotation axis, and thus the centrifugal forcehas components both perpendicular and parallel to the sample surface 240b, which defines the X-Y plane. When the bond is ruptured, thenanoswitch goes from looped (left) to unlooped (right) experiencing achange in length ΔL. This is identified by measuring the projectedchange in length in the X-Y plane ΔL_(obs)=ΔL cos θ.

The graphs of FIG. 19 depict the force and loading rate range of aspinning force system, showing that the system has abiologically-relevant dynamic force range. Panel A of FIG. 19 depicts agraph of force as a function of rotational speed calculated using fourdifferent types of beads (1 and 2.8 um Dynabeads, and 5 and 10 um silicabeads) for a spinning force system using a benchtop centrifuge. Usingthis set of beads, the spinning force system is capable of applying aforce range that spans eight orders of magnitude (10⁻⁴ to 10⁴ pN).

Panel B of FIG. 19 depicts the fastest and slowest force-loading ratesof the spinning force system measured using the Thermo ScientificHeraeus X1R Centrifuge. The fastest ramping rate of 317 g/s, shown inthe line to the left, can correspond to a 1,300 pN/s loading rate usinga large 10 um silica bead. The slowest ramping rate of 0.373 g/s, shownin the line to the right, can correspond to a 1.15 fN/s loading rateusing a small 1.0 um Dynabead (Invitrogen).

Nanoswitches Generally

The nanoswitches of this disclosure minimally comprise a scaffold orbackbone nucleic acid comprising one or more, and typically two or morebinding partners. The scaffold nucleic acid may be of any lengthsufficient to allow association (i.e., binding) and dissociation (i.e.,unbinding) of binding partners to occur, to be detected, and to bedistinguished from other events. In some instances, the scaffold nucleicacid is at least 1000 nucleotides in length, and it may be as long as20,000 nucleotides in length (or it may be longer). The scaffold nucleicacid may therefore be 1000-20,000 nucleotides in length, 2000-15,000nucleotides in length, 5000-12,000 in length, or any range therebetween.The scaffold may be a naturally occurring nucleic acid (e.g., M13scaffolds such as M13mp18). M13 scaffolds are disclosed by Rothemund2006 Nature 440:297-302, the teachings of which are incorporated byreference herein. The scaffold nucleic acid may be lambda DNA, in otherembodiments. The scaffold nucleic acid may also be non-naturallyoccurring nucleic acids such as polymerase chain reaction(PCR)-generated nucleic acids, rolling circle amplification(RCA)-generated nucleic acids, etc.

In some embodiments, the binding partners are positioned along thescaffold nucleic acid to yield loops and thus length changes that aredetectable. These may include loops that are about 40-100 base pairs, orabout 100-1000 base pairs, or about 500-5000 base pairs. The scaffoldmay be partially or fully single-stranded or partially or fullydouble-stranded. The complex may comprise varying lengths ofdouble-stranded regions.

The scaffold nucleic acid may comprise DNA, RNA, DNA analogs, RNAanalogs, or a combination thereof. In some instances, the bindingpartners are conjugated to a scaffold nucleic acid via hybridization ofoligonucleotides to the scaffold, wherein such oligonucleotides arethemselves conjugated to a binding partner. In some instances, thescaffold nucleic acid is a DNA.

The scaffold nucleic acid is hybridized to one, two or more, including aplurality, of oligonucleotides. Each of the plurality ofoligonucleotides may hybridize to the scaffold nucleic acid in asequence-specific and non-overlapping manner (i.e., each oligonucleotidehybridizes to a distinct sequence in the scaffold).

The number of oligonucleotides hybridized to a particular scaffold mayvary depending on the application. Accordingly, there may be 2 or moreoligonucleotides hybridized to the scaffold, including 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, or 1000 or more oligonucleotides.

In some instances, some oligonucleotides hybridized to the scaffoldnucleic acid will be unmodified. Unmodified oligonucleotides includeoligonucleotides that are not linked to binding partners such as bindingpartners being tested (e.g., an antibody or an antigen). In otherinstances, some or all the oligonucleotides hybridized to the scaffoldmay be modified. Modified oligonucleotides include those that are linkedto binding partners being tested (e.g., a receptor and/or its ligand, anantibody and/or its antigen, etc.). Modified oligonucleotides may alsoinclude those that are modified and thus used to immobilize thenanoswitch to a solid support such as but not limited to a bead. Suchmodified oligonucleotides including biotinylated oligonucleotides.Modified oligonucleotides may be referred to herein as “variable”oligonucleotides since these oligonucleotides may be modified by linkingto a variety of binding partners depending on the method of use.

Regions comprising scaffold hybridized to modified oligonucleotides maybe referred to herein as “variable” regions and the remaining scaffoldregions may be referred to as “fixed” regions.

The spacing of binding partners, and thus in some instances of themodified (or variable) oligonucleotides, along the length of thescaffold nucleic acid may vary. In some embodiments, the nanoswitch maycomprise three or four binding partners, and thus in some embodimentsvariable regions (e.g., three or four modified oligonucleotides). As anexample, a nucleic acid nanoswitch may comprise modifiedoligonucleotides at one or both of its ends as well as two internalmodified oligonucleotides. The modified oligonucleotides at the ends ofthe nanoswitch may be used to immobilize the nanoswitch to a solidsupport such as a bead. The modified oligonucleotides internal to thenanoswitch may be linked individually to members of a binding pair(i.e., each of the two oligonucleotides is linked to a member of thebinding pair such that the nanoswitch comprises the binding pair, witheach member of the pair on a different oligonucleotide). The internalmodified oligonucleotides may be symmetrically or quasi-symmetricallylocated around the center of the scaffold. In other words, they may bepositioned equi-distant from the center of the scaffold.

In some embodiments, the invention contemplates the use of a pluralityof nanoswitches each comprising the same binding pair. The differencebetween the nanoswitches in the plurality is the distance between thebinding pair members (i.e., the binding partners). For example, theplurality may comprise nanoswitches in which the distance between thebinding pair members is 300 base pairs, 200 base pairs, 150 base pairs,100 base pairs, 80 base pairs, 60 base pairs, and 40 base pairs. Thenanoswitches are then analyzed for their ability to form loopedstructures based on interaction between the binding partners. It isexpected that as the distance between the binding partners decreases, agreater internal force is exerted on the binding interaction.Accordingly, the binding interaction will continue until the internalforce becomes too great and the complex assumes the more energeticallyfavorable linear state. The kinetics and strength of a bindinginteraction between two binding partners can be analyzed using thisapproach.

Importantly, the distance between the binding partners will be used todistinguish association and dissociation between binding partners linkedto the nanoswitches. This is because when the binding partners areassociated with each other, a loop will be formed comprising the nucleicacid sequence that exists between the binding partners. When the bindingpartners are not associated to each other (i.e., unbound), then the loopdoes not form and the complex length is different (i.e., longer). Thenanoswitch length may be detected by direct measurements, for example,under tension, as described herein. When measured under tension, thetransition from associated to dissociated binding partners is indicatedby an increase in length of the nanoswitch.

The ability to distinguish loops of differing sizes (and thus changes oflength of different magnitudes) facilitates the use of multiplenanoswitches in a single assay where one or subsets of nanoswitches (allhaving the same loop size) are specific for a particular molecularinteraction, including for example binding to a particular analyte in asample. In these aspects, the binding partners bound to the nanoswitchdo not bind to each other but rather bind to an analyte. Accordingly, inthe presence of the analyte a loop is formed while in the absence of theanalyte no loop is formed. The looped (or closed or bound) nanoswitchhas a shorter length than the linear (or open or unbound) nanoswitch.Additionally, loops of different sizes (and thus changes in lengths) canbe distinguished from each other and as a result the presence (orabsence) of a multiple analytes (each detected by a nanoswitch having aloop of a particular size, and thus a particular change in length) canbe determined simultaneously in a multiplexed assay. Such methods may beused to detect the presence of a single or multiple analytes and mayform the basis of a diagnostic assay.

It is to be understood that several variations on the nucleic acidnanoswitches described herein. Typically, these variations all commonlycomprise a nucleic acid nanoswitches having two or more bindingpartners. The binding partners may have binding specificity for eachother or they may have binding specificity for a common analyte. Severalof the methods rely on the association and/or dissociation of bindingpartners. A change in length of the nanoswitch (e.g., from an open to aclosed conformation or from a closed to an open conformation) providesinformation about the kinetics and strength of the binding interaction.The binding partners may be non-covalently or covalently bound to thecomplex. Typically, even if the binding partners are not bound to eachother, they are nevertheless bound to the nucleic acid nanoswitch.

Thus, in a first variation, the nucleic acid nanoswitches comprises twobinding partners having binding specificity for each other. The bindingpartners are physically separate and thus spaced apart from each otheralong the length of the nanoswitch backbone (i.e., when not bound toeach other). When bound to each other, the nucleic acid nanoswitchassumes a looped (or closed or bound) conformation having a differentlength, compared to the nucleic acid nanoswitch in an open (or unbound)conformation.

In another variation, the nucleic acid complex comprises two bindingpartners having binding specificity for a common analyte. The bindingpartners are physically separate and thus spaced apart from each other(when not bound to the common analyte). When bound to the commonanalyte, the nucleic acid nanoswitch assumes a looped (or closed orbound) conformation having a different length, compared to the nucleicacid nanoswitch in an open (or unbound) conformation.

The invention further contemplates that a nucleic nanoswitches maycomprise more than two linked binding partners. The number of bindingpartners may be 2, 3, 4, 5, or more. In some embodiments, pairs ofbinding partners are provided, with each pair having binding specificityfor each other (i.e., rather than binding specificity for a commonanalyte). In some embodiments, three binding partners may be providedsuch that two binding partners compete for binding of the remainingbinding partner. The location or arrangement of the binding partners mayvary and may include serially positioned binding pairs or nested bindingpairs, or combinations thereof. As an example, assume that A1 and A2 area binding pair (e.g., first and second binding partners) and B1 and B2are a different binding pair (e.g., third and fourth binding partners),then these may be arranged as 5′-A1-A2-B1-B2-3′, or they may be arrangedas 5′-A1-B1-B2-A2-3′.

The nanoswitches comprise binding partners such as for example anantibody or an antigen. The linkage between the nucleic acid and thebinding partner may be covalent or non-covalent depending on thestrength of binding required for a particular application. They may begenerated by first incorporating a reactive group (or moiety) into thenucleic acid (or into an oligonucleotide hybridized to the nucleicacid), and then reacting this group (or moiety) with the binding partnerof interest which may or may not be modified itself. Suitable reactivegroups are known in the art. Examples of reactive groups that cancovalently conjugate to other reactive groups (leading to anirreversible conjugation) include but are not limited to amine groups(which react to, for example, esters to produce amides), carboxylicacids, amides, carbonyls (such as aldehydes, ketones, acyl chlorides,carboxylic acids, esters and amides) and alcohols. Those of ordinaryskill in the art will be familiar with other “covalent” reactive groups.Examples of reactive groups that non-covalently conjugate to othermolecules (leading to a reversible conjugation) include biotin andavidin or streptavidin reactive groups (which react with each other),antibody (or antibody fragment) reactive groups and antigens, receptorsand receptor ligands, aptamers and aptamer ligands, nucleic acids andtheir complements, and the like. Virtually any reactive group isamenable to the methods of the invention, provided it participates in aninteraction of sufficient affinity to prevent dissociation of thebinding partner from the nucleic acid nanoswitch.

It is to be understood that the scaffold nucleic acid and if used theoligonucleotides may be DNA or RNA in nature, or some combinationthereof, or some analog or derivative thereof. The term nucleic acidrefers to a polymeric form of nucleotides of any length, includingdeoxyribonucleotides, ribonucleotides, or analogs thereof. In someembodiments, the nucleic acids will be DNA in nature, and may optionallycomprise modifications at their 5′ end and/or their 3′ end.

In some embodiments, the binding partners may include without limitationantibodies (or antibody fragments) and antigens, receptors and ligands,aptamers and aptamer receptors, nucleic acids and their complements, andthe like. This list is not intended to be limited or exhaustive andother binding partners will be apparent and may be used in conjunctionwith the nanoswitches described herein.

Example

Tethered particle motion analysis was carried out for tethered beadsprior to the rupture force measurement. The lateral fluctuations of eachbead were analyzed, calculating the root mean square of thedrift-subtracted displacement and the symmetry ratio, followingpreviously established methods³³. Here, the symmetry ratio wascalculated as the square root of the ratio between the minimum andmaximum eigenvalues of the covariance matrix for the in-planedisplacement. The in-plane position of each bead was recorded for 10seconds at an acquisition rate of 10 Hz.

It should be understood that the foregoing description is intendedmerely to be illustrative thereof and that other embodiments,modifications, and equivalents are within the scope of the presentdisclosure recited in the claims appended hereto. Further, although eachembodiment described above includes certain features, the presentdisclosure is not limited in this respect. Thus, one or more of theabove-described or other features of the implantable device or methodsof use, may be employed singularly or in any suitable combination, asthe present disclosure and the claims are not limited to a specificembodiment.

REFERENCES

-   1. Bustamante C, Cheng W, Meija Y X. Revisiting the Central Dogma    One Molecule at a Time. Cell 144, 480-497 (2011).-   2. Neuman K C, Nagy A. Single-molecule force spectroscopy: optical    tweezers, magnetic tweezers and atomic force microscopy. Nat Methods    5, 491-505 (2008).-   3. Ritort F. Single-molecule experiments in biological physics:    methods and applications. J Phys-Condens Mat 18, R531-R583 (2006).-   4. Bustamante C J, Kaiser C M, Maillard R A, Goldman D H, Wilson C    A M. Mechanisms of Cellular Proteostasis: Insights from    Single-Molecule Approaches. Annual Review of Biophysics, Vol 43 43,    119-140 (2014).-   5. Greenleaf W J, Woodside M T, Block S M. High-resolution,    single-molecule measurements of biomolecular motion. Annual review    of biophysics and biomolecular structure 36, 171 (2007).-   6. Sitters G, Kamsma D, Thalhammer G, Ritsch-Marte M, Peterman E J    G, Wuite G J L. Acoustic force spectroscopy. Nat Methods 12, 47-50    (2015).-   7. Soltani M, et al. Nanophotonic trapping for precise manipulation    of biomolecular arrays. Nat Nanotechnol 9, 448-452 (2014).-   8. De Vlaminck I, et al. Highly Parallel Magnetic Tweezers by    Targeted DNA Tethering. Nano Letters 11, 5489-5493 (2011).-   9. Fazio T, Visnapuu M L, Wind S, Greene E C. DNA curtains and    nanoscale curtain rods: High-throughput tools for single molecule    imaging. Langmuir 24, 10524-10531 (2008).-   10. Ribeck N, Saleh O A. Multiplexed single-molecule measurements    with magnetic tweezers. Rev Sci Instrum 79, (2008).-   11. Kim S J, Blainey P C, Schroeder C M, Xie X S. Multiplexed    single-molecule assay for enzymatic activity on flow-stretched DNA.    Nat Methods 4, 397-399 (2007).-   12. Otten M, et al. From genes to protein mechanics on a chip.    Nature methods 11, 1127-1130 (2014).-   13. Chiou P Y, Ohta A T, Wu M C. Massively parallel manipulation of    single cells and microparticles using optical images. Nature 436,    370-372 (2005).-   14. Evans E. Probing the relation between force-lifetime-and    chemistry in single molecular bonds. Annual review of biophysics and    biomolecular structure 30, 105-128 (2001).-   15. Kim J, Zhang C-Z, Zhang X, Springer T A. A mechanically    stabilized receptor-ligand flex-bond important in the vasculature.    Nature 466, 992-995 (2010).-   16. Li P T, Bustamante C, Tinoco I. Unusual mechanical stability of    a minimal RNA kissing complex. Proceedings of the National Academy    of Sciences 103, 15847-15852 (2006).-   17. Halvorsen K, Schaak D, Wong W P. Nanoengineering a    single-molecule mechanical switch using DNA self-assembly.    Nanotechnology 22, (2011).-   18. Halvorsen K, Wong W P. Massively parallel single-molecule    manipulation using centrifugal force. Biophysical journal 98,    L53-L55 (2010).-   19. Harvey E N, Loomis A L. A microscope-centrifuge. Science 72,    42-44 (1930).-   20. Oiwa K, Chaen S, Kamitsubo E, Shimmen T, Sugi H. Steady-state    force-velocity relation in the ATP-dependent sliding movement of    myosin-coated beads on actin cables in vitro studied with a    centrifuge microscope. Proceedings of the National Academy of    Sciences 87, 7893-7897 (1990).-   21. Koussa M A, Halvorsen K, Ward A, Wong W P. DNA nanoswitches: a    quantitative platform for gel-based biomolecular interaction    analysis. Nat Methods 12, 123-U148 (2015).-   22. Cheng W, Arunajadai S G, Moffitt J R, Tinoco I, Bustamante C.    Single-base pair unwinding and asynchronous RNA release by the    hepatitis C virus NS3 helicase. Science 333, 1746-1749 (2011).-   23. Yu Z, et al. Tertiary DNA structure in the single-stranded hTERT    promoter fragment unfolds and refolds by parallel pathways via    cooperative or sequential events. Journal of the American Chemical    Society 134, 5157-5164 (2012).-   24. Baumann C G, Smith S B, Bloomfield V A, Bustamante C. Ionic    effects on the elasticity of single DNA molecules. P Natl Acad Sci    USA 94, 6185-6190 (1997).-   25. Lee C H, Danilowicz C, Conroy R S, Coljee V W, Prentiss M.    Impacts of magnesium ions on the unzipping of gimel-phage DNA. J    Phys-Condens Mat 18, S205-S213 (2006).-   26. Mao H, Arias-Gonzalez J R, Smith S B, Tinoco I, Bustamante C.    Temperature control methods in a laser tweezers system. Biophysical    journal 89, 1308-1316 (2005).-   27. Williams M C, Wenner J R, Rouzina I, Bloomfield V A. Entropy and    heat capacity of DNA melting from temperature dependence of single    molecule stretching. Biophysical Journal 80, 1932-1939 (2001).-   28. Stephenson W, et al. Combining temperature and force to study    folding of an RNA hairpin. Physical Chemistry Chemical Physics 16,    906-917 (2014).-   29. Danilowicz C, Kafri Y, Conroy R S, Coljee V W, Weeks J,    Prentiss M. Measurement of the phase diagram of DNA unzipping in the    temperature-force plane. Physical Review Letters 93, (2004).-   30. Rief M, Clausen-Schaumann H, Gaub H E. Sequence-dependent    mechanics of single DNA molecules. Nature Structural & Molecular    Biology 6, 346-349 (1999).-   31. Zhang X, Halvorsen K, Zhang C-Z, Wong W P, Springer T A.    Mechanoenzymatic cleavage of the ultralarge vascular protein von    Willebrand factor. Science 324, 1330-1334 (2009).-   32. Lipfert J, Kerssemakers J W J, Jager T, Dekker N H. Magnetic    torque tweezers: measuring torsional stiffness in DNA and RecA-DNA    filaments. Nat Methods 7, 977-U954 (2010).-   33. Nelson P C, Zurla C, Brogioli D, Beausang J F, Finzi L,    Dunlap D. Tethered particle motion as a diagnostic of DNA tether    length. The Journal of Physical Chemistry B 110, 17260-17267 (2006).-   34. Danilowicz, C. et al. Measurement of the phase diagram of DNA    unzipping in the temperature-force plane. Phys Rev Lett 93, doi:Doi    10.1103/Physrevlett.93.078101 (2004).-   35. De Vlaminck, I. & Dekker, C. Recent advances in magnetic    tweezers. Annual review of biophysics 41, 453-472 (2012).-   36. Halvorsen, K., Schaak, D. & Wong, W. P. Nanoengineering a    single-molecule mechanical switch using DNA self-assembly.    Nanotechnology 22, doi:Doi 10.1088/0957-4484/22/49/494005 (2011).-   37. Koussa, M. A., Halvorsen, K., Ward, A. & Wong, W. P. DNA    nanoswitches: a quantitative platform for gel-based biomolecular    interaction analysis. Nat Methods 12, 123-U148, doi:Doi    10.1038/Nmeth.3209 (2015).

What is claimed is:
 1. An apparatus for measuring a characteristic of asample, the apparatus being a spinning force system, and comprising: amodule comprising: a sample holder; a light source configured toilluminate the sample; an imaging axis oriented along a direction atwhich light from the light source hits the sample holder; and a detectorconfigured to receive light from the light source, wherein the module issized and dimensioned to fit within a centrifuge receptacle having avolume of less than or equal to 1 L, and wherein the spinning forcesystem is arranged such that a direction of centrifugal force applied tothe sample holder by a centrifuge and the imaging axis are misaligned inorder to track lateral particle motion.
 2. The apparatus of claim 1,wherein the volume is of less than or equal to 400 mL.
 3. The apparatusof claim 1, wherein the sample holder, light source and detector arephysically misaligned.
 4. The apparatus of claim 3, wherein modulefurther comprises at least one mirror that directs light from the lightsource to the sample holder or to the detector.
 5. The apparatus ofclaim 3, wherein the sample holder and light source are aligned along aline and the detector is misaligned from the line.
 6. The apparatus ofclaim 5, wherein the module forms a U-shape having a first leg and asecond leg.
 7. The apparatus of claim 6, wherein the sample holder andlight source are positioned within the first leg and the detector ispositioned within the second leg.
 8. The apparatus of claim 6, whereinthe first leg is longer than the second leg.
 9. The apparatus of claim1, wherein the module further comprises an objective.
 10. The apparatusof claim 9, wherein the sample holder, light source and objective arealigned along a line and the detector is misaligned from the line.
 11. Amethod comprising: attaching a particle to a surface through a molecularinteraction associated with a first molecule and a second molecule;rotating the surface about an axis of rotation to apply a centrifugalforce to the particle, the centrifugal force having a direction; hittingthe particle with light from a light source; detecting an image of theparticle with a detector during rotation of the surface, the imagecontaining information representing a characteristic of the molecularinteraction; and determining the characteristic of the molecularinteraction based on the detected image, wherein an imaging axis isangled relative to the direction of the centrifugal force, the imagingaxis being oriented along the direction at which light from the lightsource hits the particle to permit tracking of lateral particle motion.12. The method of claim 11, wherein the surface is held within acentrifugal bucket, and the bucket is at an oblique angle relative tothe axis of rotation.
 13. The method of claim 11, wherein: the step ofattaching a particle to a surface comprises attaching a nucleic acidnanoswitch to the surface, wherein the nucleic acid nanoswitch comprisesa nucleic acid conjugated to the particle on its free end.
 14. Themethod of claim 11, further comprising: inserting the surface into acentrifuge; and selecting a centrifuge temperature using a temperaturecontrol built into the centrifuge.
 15. The method of claim 14, furthercomprising: changing the centrifuge temperature using the temperaturecontrol built into the centrifuge.
 16. The method of claim 11, wherein:the step of rotating the surface about the axis of rotation comprisesrotating the surface a first time; after rotating the surface the firsttime, stopping rotation of the surface; and after stopping rotation ofthe surface, rotating the surface a second time to apply a force to theparticle; the step of detecting an image of the particle comprisesdetecting images of the particle during rotation of the surface thefirst time and the second time, the images containing informationrepresenting a characteristic of the molecular interaction; and the stepof determining the characteristic of the molecular interaction comprisesdetermining the characteristic of the molecular interaction based on thedetected images.
 17. The apparatus of claim 1, further comprising thecentrifuge.